Project description:We recently developed an easy, efficient and scalable method for tagging and live cell imaging of non-repetitive, endogenous chromosome regions via CRISPR/Cas9 mediated knock-in of a TetO repeat. For this purpose, we created optimized and irregular 48-mer and 96-mer TetO repeats. Since it is known that repetitive regions in the human genome can induce H3K9me3-mediated heterochromatin formation, we tested whether 48-mer and/or 96-mer TetO repeats induce H3K9me3 flanking their insertion sites. Using a newly developed method called as CUT&RUN, we showed that there was no significant difference in the H3K9me3 pattern flanking the insertion sites of TetO repeats when compared to wild-type cells.
Project description:Retroelement activation is emerging as a significant factor in the pathogenesis of neurodegenerative diseases. SINE-VNTR-Alu (SVAs) are hominid-specific retroelements that create genetic variation through insertion polymorphisms and variable short tandem repeat (STR) lengths. We investigate how the SVA (CCCTCT)n STR contributes to the striatal neurodegenerative disorder X-linked Dystonia Parkinsonism (XDP), where the repeat expansion length within the pathogenic SVA is inversely correlated with disease onset age. Phenotypic and transcriptomic analysis of XDP and isogenic SVA-deleted striatal organoids revealed that the SVA insertion drives hallmarks of neurodegeneration, including transcriptional dysregulation, decreased neuronal activity, and apoptosis, which are ameliorated by SVA deletion. We identify a (AGAGGG)n hexamer-containing RNA in the SVA that increases expression during organoid maturation and drives R-loop formation in organoids and XDP brain tissue. Knockdown of the (AGAGGG)n hexamer-containing RNA by antisense oligonucleotides rescues apoptosis in the XDP organoids. We demonstrate that a retrotransposon-derived tandem repeat RNA could cause neurodegeneration.
Project description:CD209L is a membrane glycoprotein with known glycan-binding properties and it also contains 2 N-glycosylation sequons at sites N92 and N361. Treatment with PNGase F in the presence of H218O, which removes N-linked glycans and isotopically labels the formerly-glycosylated site, confirmed that both unmodified and formerly-glycosylated versions of the peptide spanning the N92 N-glycosylated sequon were present. This suggests that a portion of CD209L protein is N-glycosylated at site N92. nUPLC-MS/MS analyses of CD209L digests enabled detection of a glycopeptide consistent with high-mannose type N-linked glycosylation.
Project description:To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications.
Project description:Dominantly inherited expanded repeat neurodegenerative diseases are typically caused by the expansion of existing variable copy number tandem repeat sequences in otherwise unrelated genes. Repeats located in translated regions encode polyglutamine that is thought to be the toxic agent, however in several instances the expanded repeat is in an untranslated region, necessitating multiple pathogenic pathways or an alternative common toxic agent. As numerous clinical features are shared by several of these diseases, and expanded repeat RNA is a common intermediary, RNA has been proposed as a common pathogenic agent. Various forms of repeat RNA are toxic in animal models, by multiple distinct pathways. In Drosophila, repeat-containing double-stranded RNA (rCAG.rCUG~100) toxicity is dependent on Dicer processing evident with the presence of single-stranded rCAG7, which have been detected in affected HD brains. Microarray analysis of Drosophila rCAG.rCUG~100 repeat RNA toxicity revealed perturbation of several pathways including innate immunity. Recent reports of elevated circulating cytokines prior to clinical onset, and age-dependent increased inflammatory signaling and microglia activation in the brain, suggest that immune activation precedes neuronal toxicity. Since the Toll pathway is activated by certain forms of RNA, we assessed the role of this pathway in RNA toxicity. We find that rCAG.rCUG~100 activates Toll signaling and that RNA toxicity is dependent on this pathway. The sensitivity of RNA toxicity to autophagy further implicates innate immune activation. Expression of rCAG.rCUG~100 was therefore directed in glial cells and found to be sufficient to cause neuronal dysfunction. Non-autonomous toxicity due to expanded repeat-containing double-stranded RNA mediated activation of innate immunity is therefore proposed as a candidate pathway for this group of human genetic diseases. The heads from newly eclosed male Drosophila were used for RNA extraction and profiling on Affymetrix Drosophile Genome 2.0 microarrays. Nine samples were analysed, representing control and experimental lines. Two independent lines of rCAG.rCUG~100 double-stranded RNA were analysed in triplicate. These were compared to 4xUAS control analysed in triplicate. All transgenes were expressed using the elavII-GAL4 pan-neuronal driver. Candidates were selected from the pool of transcripts which showed a 'present' call in all samples. T-tests were performed on raw values to determine samples that showed a significant difference with a P-value < 0.05.
Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensinM-bM-^@M-^Ys HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain. Analysis of condensin binding genomewide in a wild type and a condensin mutant